Sequencing and modeling of anti-DNA immunoglobulin Fv domains. Comparison with crystal structures

Models for the three-dimensional structures of the combining regions of six DNA-binding antibodies have been derived from the sequence data for their Fv domains presented here. Using the amino acid sequences and the canonical structure classes described by Chothia and Lesk (Chothia, C., and Lesk, A.M. (1987) J. Mol. Biol. 196, 901), model loops were selected from immunoglobulin domains of known structure for five of the six antibody hypervariable regions. Models for the third complementarity-determining region of the heavy chain were constructed from known immunoglobulin loops of similar length and sequence. Comparison of three of the models with the respective crystal structure indicates that this procedure can generate a working model of the antibody combining region that provides useful information on the nature of the interactions between antibodies and nucleic acids. As part of our continuing investigation into the structural basis of antibody-DNA recognition, the observed and predicted models for the combining regions of nucleic acid-binding antibodies have been examined. In general, single strand-specific antibodies have deep clefts where the antigen might bind, whereas duplex-specific antibodies present a relatively flat surface. In addition, on the basis of both sequence and structure, there is little to distinguish autoimmune antibodies from those produced by immunization. Testable hypotheses for how these antibodies might interact with single- and double-stranded nucleic acids are presented.     

Lipomatous hypertrophy of the atrial septum: diagnosis with fat suppressed MR imaging

We present a case of a 50-year-old female evaluated for a 1-year history of numbness of the first and second toe of the right foot. Echocardiography performed in order to exclude cardiovascular compromise revealed a granular mass originating from the posterior part of the interatrial septum. On subsequent magnetic resonance imaging (MRI) with fat suppression sequences, the final diagnosis of lipomatous hypertrophy of the interatrial septum, a benign and underrecognized condition characterized by septal accumulation of fatty tissue, was made. Although no previous reports have focused on this, tailored cardiac MR with fat suppression sequences proved to be an excellent noninvasive method in assessing an accurate diagnosis and in differentiating lipomatous hypertrophy of the atrial septum from other cardiac neoplasms.     

Schwannoma with sweat duct differentiation

In a randomized trial of second line hormone therapy 56 postmenopausal women with advanced breast cancer received low dose aminoglutethimide (AGT) 125 mg twice daily without hydrocortisone (27 patients), or hydrocortisone (HC) 20 mg twice daily (29 patients). The two groups were well-matched for previous response to tamoxifen (TAM) therapy (AGT (35%) vs HC (32%)) and for relapse on adjuvant TAM. The mean age of the two groups was 69.2 years (AGT) and 63.2 years (HC). Liver metastases were present in 29% (AGT) and 33% (HC). The response rates were 11% (AGT) and 21% (HC). At 12 months the failure of treatment rate was 80% (AGT) and 70% (HC). Survival at 12 months was 50% for both groups. At 12 months 5/12 survivors were still on AGT and 8/12 on HC. These preliminary findings have so far failed to show any statistical difference in tumour response, time to treatment failure or survival between low-dose AGT and HC as second-line hormone treatment post-tamoxifen in advanced breast cancer.     

Decisional balance and stage of change for adolescent drinking

The Transtheoretical Model of Change has been proven very effective in explaining both the acquisition and cessation of many health related behaviors. In this study, this model was applied to the domain of immoderate alcohol use among adolescents (usually drinking three or more drinks per occasion). Measures for two constructs of the model were developed: Stage of Change and Decisional Balance. A total of 853 tenth and eleventh graders who attend vocational training programs were administered a 37-item decisional balance questionnaire and a 5-item staging measure. A short (16-item) psychometrically sound Decisional Balance Inventory was developed based on an exploratory factor analysis that identified two factors, the Pros and Cons of Alcohol Use. The factor structure was confirmed using structural modeling techniques on a hold-out sample. Based on a combination of model fit and parsimony considerations, an uncorrelated model was selected (IFI2 = .909). Students were classified into one of nine stages of acquisition or cessation. External validity was established by the significant and meaningful differences between the stages of change on the pros and cons of alcohol use. Implications of this research are discussed.     

Bioactivation and irreversible binding of the cognition activator tacrine using human and rat liver microsomal preparations. Species difference

Tacrine's [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, (THA)] metabolic fate was examined using human and rat liver microsomal preparations. Following 1-hr incubations with human microsomes, [14C]THA (0.4 microM) was extensively metabolized to 1-hydroxyTHA with trace amounts of 2-, 4-, and 7-hydroxyTHA also produced. Poor recovery of radioactivity in the postreaction incubates suggested association of THA-derived radioactivity with precipitated microsomal protein. After exhaustive extraction, 0.034, 0.145, 0.126, and 0.012 nmol eq bound/mg protein/60 min of THA-derived radioactivity was bound to human liver preparations H109, H111, H116, and H118, respectively. Preparations H109 and H118 were lower in P4501A2 content and catalytic activity as compared with preparations H111 and H116. Incubations of equimolar [14C]1-hydroxyTHA with human liver microsomes also resulted in binding to protein, although to a lesser extent than observed with THA. [14C]THA (0.4 microM) was incubated for 1 hr with rat liver microsomes (1 microM P-450) prepared from noninduced (N), phenobarbital (PB), isoniazid (I), and 3-methylcholanthrene (3-MC)-pretreated animals. In all incubations, 1-hydroxyTHA was the major biotransformation product detected. After exhaustive extraction, 0.048, 0.054, 0.049, and 0.153 nmol eq/mg protein/60 min of THA-derived radioactivity was bound to microsomal protein from N, PB, I, and 3-MC pretreated rats. Increased binding with 3-MC induced rat liver preparations suggests the involvement of the P-450 1A subfamily in THA bioactivation. Glutathione (5 mM) coincubation inhibited the irreversible binding of THA-derived radioactivity in both human and 3-MC-induced rat liver preparations, whereas human epoxide hydrase (100 micrograms/incubate) had a relative minor effect. A mechanism is proposed involving a putative quinone methide(s) intermediate in the bioactivation and irreversible binding of THA. A species difference in THA-derived irreversible binding exists between human and noninduced rat liver microsomes, suggesting that the rat is a poor model for studying the underlying mechanism(s) of THA-induced elevations in liver marker enzymes found in clinical investigations.     

Insulin-like growth factors (IGF) I and II and IGF binding proteins 1, 2 and 3 during low-dose growth hormone (GH) infusion and sequential euglycemic and hypoglycemic glucose clamps: studies in GH-deficient patients

To evaluate the short-term effects of growth hormone (GH), insulin and different levels of glycemia on insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 1, 2 and 3, we studied six GH-deficient adolescents during a night and the following day in the postabsorptive (basal) state followed by sequential euglycemic (5 mmol/l) and hypoglycemic (3 mmol/l) glucose clamps concomitant with an intravenous infusion (starting at 24.00 h) of GH (35 micrograms/h) or saline. Current GH therapy was withdrawn 24 h prior to each study. Nocturnal levels of IGF-I, IGF-II, IGFBP-2 and IGFBP-3 remained stable during both studies. Nocturnal serum IGFBP-1 increased and correlated inversely with insulin in both studies. Regression analysis revealed a significant inverse correlation between mean nocturnal IGFBP-2 and IGFBP-3 levels. During the daytime, serum IGF-I declined slowly during saline infusion, whereas serum IGF-II remained stable in both studies. Serum IGFBP-1 displayed a gradual significant decline during the basal state and the euglycemic and hypoglycemic clamps seemed to be unaffected by GH levels. By contrast, serum IGFBP-2 remained stable during the same period in both the GH and the saline study. Serum IGFBP-3 declined insignificantly during the daytime in the saline study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes

Selected properties of Caco-2 cells were examined after disparate transepithelial electrical resistance (TEER) measurements were observed in two populations of Caco-2 cells. Comparisons were made between the early passages of Caco-2 cells (Caco-2E, passages 35-47) and the later passages of cells (Caco-2L, passages 87-112). Transmission electron microscopy revealed that regions of Caco-2L cells were composed of multiple cell layers rather than the monolayers observed in Caco-2E cells. Epithelial cell height (or barrier thickness) was not significantly different between the two cell populations. Intercellular and intracellular lumina were observed in the Caco-2L cells, but not in the Caco-2E cells. Results of [3H]thymidine incorporation assays showed significantly higher cell proliferation rates in Caco-2L cells relative to Caco-2E cells. Despite morphological and physiological changes, there were no significant differences in the apparent permeabilities for D-mannitol (paracellular diffusion marker), hydrocortisone (transcellular diffusion marker), or dipeptide, Gly-Sar (carrier-mediated transcellular transport marker) between the two populations of cells. The higher TEER values in Caco-2L cells may be the results of a slight perturbation of tight junctions associated with both the multiple cell layers and the presence of intercellular lumina.     

Involvement of donor T-cell cytotoxic effector mechanisms in preventing allogeneic marrow graft rejection

Donor CD8 cells play a pivotal role in preventing allogeneic marrow graft rejection, possibly by generating cytotoxic effectors needed to eliminate recipient T cells remaining after the pretransplant conditioning regimen or by producing cytokines needed to support the growth and differentiation of hematopoietic stem cells. In the present study, we assessed the role of donor T-cell cytotoxic effector function as a mechanism for eliminating recipient CD8 cells that cause marrow graft rejection in mice. The ability to prevent rejection was minimally affected by the presence of a defect in Fas ligand binding or by the absence of granzyme B but was severely affected by the absence of perforin. Doubly mutant perforin-deficient, Fas ligand-defective CD8 cells were completely unable to prevent rejection. Our results indicate first that recipient CD8 effectors responsible for causing marrow graft rejection are sensitive to cytotoxicity mediated by both perforin- and Fas-ligand-dependent mechanisms, and second that donor T cells must have at least one functional cytotoxic mechanism to prevent allogeneic marrow graft rejection.